Characterization of Novel Calmodulin Binding Domains within IQ Motifs of IQGAP1

다국어 초록 (Multilingual Abstract)

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca^(2+)-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAG-hCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca^(2+)-independent or -depen- dent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca^(2+)/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca^(2+)/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca^(2+)-dependent or -independent manner.

참고문헌 (Reference)

1 Mateer, S.C., "The mechanism for regulation of the F-actin binding activity of IQGAP1 by calcium/calmodulin" 277 : 12324-12333, 2002

2 Sung Cheol Koo, "The Calmodulin-Binding Transcription Factor OsCBT Suppresses Defense Responses to Pathogens in Rice" 한국분자세포생물학회 27 (27): 563-570, 2009

3 Rhoads, A.R., "Sequence motifs for calmodulin recognition" 11 : 331-340, 1997

4 Shen, X., "Scanning the human proteome for calmodulin-binding proteins" 102 : 5969-5974, 2005

5 Alexander, K.A., "Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein" 262 : 6108-6113, 1987

6 Baudier, J., "Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43). that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain" 266 : 229-237, 1991

7 Jang, D.J., "Proteomic and biochemical studies of calcium- and phosphorylation-dependent calmodulin complexes in Mammalian cells" 6 : 3718-3728, 2007

8 Petrova, T.V., "Phosphorylation of the IQ domain regulates the interaction between Ca2+-vector protein and its target in Amphioxus" 271 : 26646-26652, 1996

9 Blumenthal, D.K., "Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase" 82 : 3187-3191, 1985

10 White, C.D., "IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis" 583 : 1817-1824, 2009

11 Noritake, J., "IQGAP1: a key regulator of adhesion and migration" 118 : 2085-2092, 2005

12 Briggs, M.W., "IQGAP1-mediated stimulation of transcriptional co-activation by beta-catenin is modulated by calmodulin" 277 : 7453-7465, 2002

13 Roy, M., "IQGAP1 is a scaffold for mitogen-activated protein kinase signaling" 25 : 7940-7952, 2005

14 Swart-Mataraza, J.M., "IQGAP1 is a component of Cdc42 signaling to the cytoskeleton" 277 : 24753-24763, 2002

15 Ho, Y.D., "IQGAP1 integrates Ca2+/calmodulin and Cdc42 signaling" 274 : 464-470, 1999

16 Roy, M., "IQGAP1 binds ERK2 and modulates its activity" 279 : 17329-17337, 2004

17 Briggs, M.W., "IQGAP1 as signal integrator: Ca2+, calmodulin, Cdc42 and the cytoskeleton" 542 : 7-11, 2003

18 Li, Z., "IQGAP1 and calmodulin modulate E-cadherin function" 274 : 37885-37892, 1999

19 Li, Z., "Elucidation of the interaction of calmodulin with the IQ motifs of IQGAP1" 278 : 4347-4352, 2003

20 Szymanska, G., "Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase" 252 : 96-105, 1997

21 Bahler, M., "Calmodulin signaling via the IQ motif" 513 : 107-113, 2002

22 Joyal, J.L., "Calmodulin modulates the interaction between IQGAP1 and Cdc42. Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry" 272 : 15419-15425, 1997

23 Porter, J.A., "Calmodulin binding to Drosophila NinaC required for termination of phototransduction" 14 : 4450-4459, 1995

24 Mizumoto, T., "Calmodulin activities are significantly increased in both uninvolved and involved epidermis in psoriasis" 85 : 450-452, 1985

25 Ruegg, J.C., "A calmodulin-binding peptide relaxes skinned muscle from guinea-pig taenia coli" 414 : 282-285, 1989